Drosophila models of human mitochondrial diseases

人类线粒体疾病的果蝇模型

基本信息

批准号：
10756280
负责人：
NORBERT PERRIMON
金额：
$ 86.73万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10756280
关键词：
Affect Affinity Aging Alpaca Animal Model Base Sequence Biochemical Biological Assay Biological Models Biomedical Research Chimeric Proteins Cloning Clustered Regularly Interspaced Short Palindromic Repeats Code Communities Complementary DNA Defect Degenerative Disorder Detection Diabetes Mellitus Drosophila genus Drug or chemical Tissue Distribution Enhancement Technology Epitopes Exons Functional disorder Gene Structure Genes Genetic Genome engineering Heart failure Human Immunoprecipitation Individual Knock-in Liver diseases Mediating Metabolic Diseases Mitochondria Mitochondrial Diseases Mitochondrial Proteins Modeling Morphology Muscle Weakness Mutation Nerve Degeneration Organ Orthologous Gene Pathology Patients Phenotype Premature aging syndrome Protein Isoforms Proteins Protocols documentation Public Domains Publishing R24 RNA Interference Reagent Reproducibility Resource Development Resources Role Seizures Signal Transduction Technology Tertiary Protein Structure Tissues Transgenic Organisms United States National Institutes of Health Validation cost effective experimental study fly gene function human disease human model in vivo interest kidney dysfunction knock-down nanobodies response tool

项目摘要

PROJECT SUMMARY / ABSTRACT Defects in mitochondria are associated with a spectrum of conditions, ranging from metabolic and degenerative diseases to premature aging. Mitochondrial defects are also associated with multiple organ defects, including muscular weakness, cardiac failure, diabetes, renal dysfunction, and hepatic disease. One approach to understanding the physiopathology of mitochondrial diseases is to create animal models that enable detailed mechanistic studies of the underlying perturbations. Drosophila is an established cost-effective model system for studying human diseases, including mitochondrial diseases. Validation of fly models relies on two critical sets of tools: reagents that perturb gene function and reagents that recognize proteins. While perturbation reagents are readily available for nearly all 14,000 Drosophila genes, there are relatively few reagents for detection of fly proteins. Our proposal focuses on generating reagents that will serve as a resource to enable the characterization and validation of Drosophila models of human mitochondrial diseases. We will generate two sets of reagents, based on the target gene structure, for the detection of proteins encoded by 394 high confidence Drosophila orthologs of human mitochondrial disease genes. For Group 1 genes (Aim 1), where all isoforms share the same C-terminus exon, we will insert a NanoTag epitope at the C-terminus of the endogenous gene using CRISPR-based genome engineering. We selected the 3’ end to insert the tag as inserting it at the N terminus could interfere with the mitochondrial localization signal. The resulting protein fusions can then be recognized by an existing high-affinity nanobody against the NanoTag. For Group 2 genes (Aim 2), which encode multiple isoforms, including some with different C-terminus exons, we will use a protein domain common to all isoforms to screen for nanobodies. These reagents will be used together with existing RNAi transgenic lines to validate fly models of human mitochondrial diseases (Aim 3). The resource of nanobodies and fly stocks with NanoTags ‘knocked-in’ to endogenous genes will dramatically expand the scope of available reagents for detection and biochemical characterization of fly mitochondrial proteins.

项目概要/摘要线粒体缺陷与一系列病症有关，包括代谢和退行性病变线粒体缺陷也与多种器官缺陷有关，包括早衰。肌肉无力、心力衰竭、糖尿病、肾功能障碍和肝脏疾病的一种方法。了解线粒体疾病的病理生理学就是创建能够详细了解线粒体疾病的动物模型果蝇的潜在扰动的机制研究是一个已建立的具有成本效益的模型系统。用于研究人类疾病，包括线粒体疾病，苍蝇模型的验证依赖于两个关键集。工具：扰乱基因功能的试剂和识别蛋白质的试剂。几乎所有 14,000 个果蝇基因均可轻松获得，用于检测果蝇的试剂相对较少我们的建议重点是生成试剂，作为实现这一目标的资源。我们将生成人类线粒体疾病的果蝇模型的表征和验证。两套试剂，基于目标基因结构，用于检测394 high编码的蛋白质人类线粒体疾病基因的置信度果蝇直系同源物对于第 1 组基因（目标 1），其中所有同工型共享相同的 C 端外显子，我们将在内源性的 C 端插入 NanoTag 表位我们使用基于 CRISPR 的基因组工程选择了 3' 端来插入标签，因为将其插入在 N 处。末端可能会干扰线粒体定位信号。被现有的针对 NanoTag 的高亲和力纳米抗体识别（目标 2）。编码多种亚型，包括一些具有不同 C 端外显子的亚型，我们将使用共同的蛋白质结构域这些试剂将与现有的 RNAi 转基因一起使用。线来验证人类线粒体疾病的苍蝇模型（目标 3）纳米抗体和苍蝇种群的资源。与内源基因“敲入”的 NanoTags 将极大地扩大可用试剂的范围果蝇线粒体蛋白的检测和生化表征。